City College of the City University of New York Jane C. Gallagher 3/86 — 12/87

ZK-4-04136-5; ZK 4-04-136-04

The purpose of these studies was to investigate the intragenetic variability (e. g., between various isolates of a single species) in microalgae with potential for high lipid production. The rationale for this work with respect to the ASP is that variability within and between species of microalgae

has implications for algal collection strategies, for strain selection for high lipid producers, and for genetic manipulation of microalgae by classical breeding or genetic engineering.

Historically, microalgae have been classified based on morphological similarities. Previous studies by Dr. Gallagher and others (see Gallagher 1986) suggested significant physiological variability between isolates of a single species. In these studies, various isolates of a species grown under identical conditions (to control for environmentally induced changes in gene expression) often showed significant differences in characteristics such as nutrient uptake, growth rates, and pigment content. These results indicated that there may be inherent genetic differences between the individual strains. The studies by Dr. Gallagher compared electrophoretic banding patterns of specific proteins to obtain quantitative estimates of the genetic differences between isolates of two genera of oil-producing microalgae.

The organisms studied were A. coffeiformis (class Bacilliarophyceae) and Nannochloropsis spp. (class Eustigmatophyceae). The basic approach was to streak the isolated algae onto agar plates, then to pick single colonies and restreak the cells to ensure that each strain was unialgal. The isolates were propagated under identical growth conditions to minimize differences caused by environmentally induced changes in gene expression. Each strain was examined using light microscopy (LM) and scanning electron microscopy (SEM) to look for morphological differences and to confirm species identity. Crude protein extracts from each strain were separated by polyacrylamide gel electrophoresis. The gel was then stained to detect several specific enzymes, including phosphoglucose isomerase, hypoxanthine dehydrogenase, a — ketoglutarate dehydrogenase, malate dehydrogenase, a-hydroxybutarate dehydrogenase, and tetrazolium oxidase. (Dr. Gallagher also tried unsuccessfully to stain for several other enzymes. Poor staining may be a consequence of the location of these enzymes within cellular membranes in microalgae.) An extract from the diatom Skeletonema costatum (clone SKEL) was run on each gel to serve as an internal standard, and the migration pattern for each enzyme was reported as the ratio of the migration distance for the unknown Amphora or Nannochloropsis enzyme to the migration of the known enzyme from Skeletonema. An example of this type of experiment is shown in Figure III. B. 1. This method allowed for detection of very small differences in the migration patterns of the various forms of the enzymes. These differences could result from subtle variations in protein charge or conformation due to one or several amino acid changes. Isolates that showed two bands for a specific enzyme were assumed to be heterozygous at that allele.

For the studies of Amphora, Dr. Gallagher obtained 47 isolates, 32 of which were isolated from a salt marsh in Woods Hole, Massachusetts, on the same day in August 1985. Another six strains had been isolated from the same site during the summers of 1979 or 1980 and maintained in culture, and five strains were obtained from laboratory cultures maintained by other investigators. It is unclear from Dr. Gallagher’s reports how many of the 47 Amphora isolates were tested as described earlier.

All strains that were subsequently analyzed for enzyme banding patterns were first examined by LM and SEM. Microscopy confirmed that all the strains were A. coffeiformis, although some

variation was observed in the morphology of the frustule between strains, for example, in the presence or absence of costae (rib-like protrusions) or in the shape or number of punctae (holes). These changes were assumed to be due to genetic differences between the strains, as unialgal clones maintained in culture for 6-7 years did not show variations in frustule morphology between individuals. Genetic similarity was calculated based on the electrophoretic banding patterns using the statistical methods of Nei (1972). The zymograms indicated significant variation between isolates of the same species, even between strains isolated from the same site on the same day. These differences were not correlated with the extent of morphological variation, and some morphologically identical strains showed differences in the protein banding patterns.

For Nannochloropsis, 115 strains were obtained, all from culture collections. The electrophoretic banding patterns also indicated significant genetic diversity between strains, even between samples isolated from the same location. However, the zymogram data for Nannochloropsis was limited due to the high percentage of “null” alleles (no staining of some enzymes) in some isolates. It was unclear whether this was caused by undetermined genetic differences between the isolates (and between Amphora and Nannochloropsis), or due to difficulties in extraction of the proteins from Nannochloropsis. More data would be needed to fully analyze the genetic differences between isolates of this genus.

What are the implications of this research for the Aquatic Species Program? The significant amount of genetic diversity between individuals of a species, even when isolated from very similar locations, suggests that researchers involved in collecting microalgal strains as potential lipid producers should obtain more than one isolate from each site. In fact, these results suggest that it may be adequate to sample fewer sites to obtain a sufficiently varied collection of microalgal strains.

In a previous study (discussed in Gallagher 1985), Dr. Gallagher described experiments performed on isolates of the diatom S. costatum similar to those described here. The data suggested significantly less genetic variation between isolates of Skeletonema, even between strains isolated from different locations, than was seen between Amphora strains isolated from the same environment. This difference was attributed to the fact that Amphora is an attached, benthic organism that produces amoeboid gametes, whereas Skeletonema is planktonic, and produces swimming sperm. These “lifestyle” differences would result is lower potential for gene flow between Amphora populations, although the presence of heterozygotes indicates interbreeding among Amphora at localized sites. These observations suggest that benthic organisms may have greater genetic diversity than planktonic forms.

Based on the data in this study, Dr. Gallagher also concluded that breeding or genetic engineering of microalgae may be more successful using morphologically similar phenotypes, as her results suggest less diversity at the protein level between identical morphotypes. However, genetic engineering research during the past 15 years in other organisms indicates that cells can often express genes from very different species, so these differences between strains probably will not affect the expression of genes transferred between these similar organisms.

While working under the SERI subcontract, Dr. Gallagher also participated in a study that provided evidence that the carotenoid violaxanthin functions as a major light harvesting pigment in Nannochloropsis (Owens et al. 1987). Carotenoids generally are considered accessory pigments in photosynthetic organisms, involved primarily in photoprotection, fluorescence quenching, and light harvesting. Nannochloropsis is a member of the class Eustigmatophyceae, which are unusual in that they can contain violaxanthin as up to 60% of their total pigments. These authors used room temperature fluorescence excitation and emission data to provide the first evidence that violaxanthin can function in photosynthetic light harvesting.

Understanding the fundamental processes involved in microalgal photosynthesis is important to the ASP since light-driven photosynthesis results in the production of chemical reductants that drive the synthetic dark reactions; lipids are storage products that can be produced from excess photosynthate. One possible implication is that carotenoids absorb at different wavelengths than chlorophyll, absorbing green light that penetrates into the water column. This feature could be beneficial for mass culture of organisms, allowing denser cultures to grow in a deep raceway.

I Publications:

Gallagher, J. C. (1986). “Population genetics of microalgae.” In Algal Biomass Technologies: An Interdisciplinary Perspective (Barclay, W.; McIntosh, R., eds.), Beihefte zur Nova Hedwigia, Heft 83, Gebruder Borntraeger, Berlin-Stugart, pp. 6-14.

Gallagher, J. C. (1987a). “Patterns of genetic diversity in three genera of oil-producing microalgae” (abstr.), FY1987Aquatic Species Program Annual Report, (Johnson, D. A.; Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3206, p. 209.

Gallagher, J. C. (1987b). “Genetic variation in oil-producing microalgae.” FY 1986 Aquatic Species Program Annual Report, (Johnson, D. A., ed.), Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, p.331-336.

Owens, T. G.; Gallagher, J. C.; Alberte, R. S. (1987) “Photosynthetic light-harvesting function of violaxanthin in Nannochloropsis spp. (Eustigmatophyceae).” J. Phycol. 23:79-85.

I Additional References:

Nei, M. (1972) Amer. Natur. 106:283.